1. Field of the Invention
The present invention relates to the field of peptides useful for binding to integrin alpha-v-beta-6 (“αvβ6”) cell surface receptors, and also to the field of biomarkers as cancer diagnostic tools.
2. Related Art
Presented below is background information on certain aspects of the present invention as they may relate to technical features referred to in the detailed description, but not necessarily described in detail. The discussion below should not be construed as an admission as to the relevance of the information to the claimed invention or the prior art effect of the material described.
Detection of pancreatic cancer remains a high priority and effective diagnostic and therapeutic tools are needed for clinical applications. Many cancer cells over express integrin αvβ6, a cell surface receptor being evaluated as a novel clinical biomarker.
Integrins are a family of heterodimeric cell surface receptors that mediate cellular adhesion to extracellular matrix proteins and serve as bi-directional signal transducers to regulate differentiation, migration, proliferation, and cell death (1, 2). Integrin αvβ3 promotes in certain embodiments, a sequence with at least neovascularization (3, 4). However in certain cancers, other integrins, such as αvβ6, become highly over expressed on cell surfaces (2, 5, 6). Therefore, this biomarker is being validated for detection of colon, liver, ovarian, pancreatic, and squamous cell cancers (7-9). Molecular imaging of integrin αvβ6 may be used to gauge receptor expression levels to determine prognosis and guide therapy (10-12). As described below, potent integrin αvβ6 binders for use with radiotracers for early cancer detection were developed.
Several integrin αvβ6 binders have been previously identified from natural and combinatorial sources. Linear αvβ6-binding peptides derived from the coat protein of foot-and-mouth-disease viruses (FMDV) generally suffered poor in vivo stability, which raise concerns about their potential immunogenicity. 64Cu-labeled versions of FMDV peptides demonstrated extremely high renal retention, which suggests that these peptides may not be ideal translational candidates. An alternative to radio-metals is the use of radio-halogens (7, 9, 13). Phage display systems have identified several linear and disulfide-cyclized peptides that bind αvβ6 (14, 15). In one study, a radio-iodinated linear peptide, HBP-1, showed rapid degradation in serum (16). One-bead-one-compound libraries have identified many binders, of which 43 18F-labeled linear peptides were tested in a high throughput live animal imaging survey (17). While some of these peptides showed promising small animal data, linear peptides or even simple disulfide-bonded peptides with stability problems may discourage translation (17). Peptide fragments can be highly immunogenic, thus rendering the parent peptide untranslatable (18). For these reasons, the present invention was developed to generate high-affinity binders that are very stable in physiological media, demonstrate low off target accumulation and effectively detect cancer in living subjects.
Engineered cystine knot peptides (knottins) have shown promise for cancer imaging with αvβ3 as a target (19-21). The cystine knot is a rigid molecular scaffold of 3-4 kDa that owes its exceptionally-high stability to three interwoven disulfide bonds and a centrally-located beta sheet. Potent receptor-binding activities have been engineered into the scaffold's solvent exposed loops (22). The knotted structure helps to resist degradation/denaturation in hostile biological, chemical and physical environments such as strong acids and boiling water (23). Cystine knots have shown exceptional structural stability during prolonged incubation in serum (19).
Moreover, their use in humans as uterogenics has not led to reports of adverse side effects, albeit without formal published studies (24, 25). Binding potency remains high for engineered knots that were subjected to long-term storage (>1 year) in water at 4° C. or stored in lyophilized form at room temperature. The N-terminus provides a sole primary amine for site-specific conjugation of imaging labels, bioactive cargo, or pharmacokinetic stabilizers. Collectively, these characteristics bode well for clinical translation.
3. Specific Patents and Publications
US Published Application 2009/0257952, entitled “Engineered Integrin binding Peptides,” by Cochran, et al., discloses engineered binding peptides comprised in EETI-II, AgRP, mini-AgRP, agatoxin or miniagatoxin scaffolds. The peptides specifically bind to integrins αvβ5 and αvβ3 and have an integrin binding XXRGDXXXX sequence. The publication discloses a randomized library of RGD mimic sequences based on different scaffolds. The publication further discloses imaging of various cancers using compounds of the invention and discusses their properties for use as imaging probes.
“Developing New Tools for the in vivo Generation/Screening of Cyclic Peptide Libraries. A New Combinatorial Approach for the Detection of Bacterial Toxin Inhibitors,” a research report from Lawrence Livermore Laboratory, published online, UCRL-TR-227590, describes synthesis of MCoTI-II.
Sommerhoff et al., “Engineered Cystine Knot Miniproteins as Potent Inhibitors of Human Mast Cell Tryptase β,” J. Mol. Biol. 395:167-175 (8 Jan. 1010) discloses inhibitors derived from a linear variant of the cyclic cystine knot miniprotein MCoTI-II, originally isolated from the seeds of Momordica cochinchinensis. A synthetic cyclic miniprotein was prepared that bears additional positive charge in the loop connecting the N- and C-termini.
Kraft S, Diefenbach B, Mehta R, Jonczyk A, Luckenbach G A, Goodman S L., “definition of an unexpected ligand recognition motif for alpha-v-beta-6 integrin,” J Biol Chem 1999; 274: 1979-85 discloses the recognition profiles of recombinant alpha-v-beta-6 and alpha-v-beta-3 integrins by using phage display screening employing 7-mer and 12-mer peptide libraries. As predicted, phages binding strongly to alpha-v-beta-3 contained ubiquitous RGD sequences. However, on alpha-v-beta-6, in addition to RGD-containing phages, one-quarter of the population from the 12-mer library contained the distinctive consensus motif DLXXL. A synthetic DLXXL peptide, RTDLDSLRTYTL (SEQ ID NO: 1), selected from the phage sequences (clone-1) was a selective inhibitor of RGD-dependent ligand binding to alpha-v-beta-6 in isolated receptor assays (IC50=20 nM), and in cell adhesion assays (IC50=50 microM).
Dicara et al., “Structure-function analysis of Arg-Gly-Asp helix motifs in alpha v beta 6 integrin ligands,” J Biol Chem. 2007 Mar. 30; 282(13):9657-65. Epub 2007 Jan. 23, discloses physical requirements for high affinity binding of ligands to alpha-v-beta-6. By combining a series of structural analyses with functional testing, the authors show that 20-mer peptide ligands, derived from high affinity ligands of alpha-v-beta-6 (foot-and-mouth-disease virus, latency associated peptide), have a common structure comprising an Arg-Gly-Asp motif at the tip of a hairpin turn followed immediately by a C-terminal helix.